There are continuing advances in the ability to modify cells. One of the most exciting areas for the treatment of genetic diseases is gene therapy. In those situations where cells can be isolated, which are relatively long-lived and can be grown in culture and expanded, one has the opportunity to isolate such cells from a host, either the diseased host or an allogeneic host. The cells may then be grown in culture, expanded, and modified by homologous recombination, and the modified cells restored to the host.
In other situations, one wishes to produce products which are exogenous to the host. In this situation, one may modify embryonic stem cells, add them to a blastocyst, and then produce a chimeric host. This chimeric host may then be mated and the progeny either used as heterozygous mutants or further mated to produce homozygous mutants. By providing for appropriate changes in the embryonic cells, one can produce various human proteins in bovine milk and the like.
Alternatively, one may wish to change the nature or characteristics of a product of a host, such as meat, vegetable oils, size, or the like. In many cases, homologous recombination will be required with both genes, since the host will be diploid. This means that one must go through an extensive number of steps in first modifying one gene, ensuring that the proper modification has occurred, then using the modified cells to repeat the process and modify the second gene. Even where the modification is a knock-out, namely introduction of an interfering sequence and/or deletion into the target locus, the process can be time-consuming and labor-intensive. There is, therefore, substantial interest in being able to develop techniques which allow for improvements in the efficiency of achieving genetic modifications at target loci.
Relevant Literature
Procedures for the modification of ES cells and detection of the modified cells may be found in Mortensen, et al., Proc. Natl. Acad. Sci. (USA) 88, 7036-7040 (1991). Paludan, et al., Gene 85, 421-426 (1989) reports enhanced resistance of cells comprising multiple neo genes. Spontaneous production of homozygous cells from heterozygous cultured cells have been reported by Nelson, et al., Mol. Cell. Biol. 9, 1284-1288 (1989); Potter, et al., Proc. Natl. Acad. Sci. (USA). 84, 1634-1637 (1987); Rajan, et al., J. Immunol, 145, 1598-1602 (1990) and Campbell, et al., Mol. Cell. Biol. 1, 336-346 (1981).